Cell therapy for the treatment of neurodegeneration

ABSTRACT

Methods are described for the isolation and selection of a heterogeneous bone marrow cell population, called NCS-01, that is effective at treating neurodegeneration. For example, NCS-01 cells are shown to treat neurodegeneration caused by ischemia. In vivo studies demonstrate that selected NCS-01 cell populations treat neurodegeneration in a standard rat middle cerebral artery occlusion (MCAO) animal model under conditions of transient or permanent total arterial occlusion. These studies also disclose that when the neurodegeneration is caused by ischemic stroke, combining the administration of a selected NCS-01 cell population with thrombolytic agents and/or mechanical methods of clot removal leads to a decrease in the volume of infarction caused by acute onset neurodegeneration. The disclosed cell therapy promises to make a significant clinical impact on patient survival after stroke.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.15/662,347 (now allowed), filed Jul. 28, 2017 which is a Continuation ofU.S. application Ser. No. 13/819,639, filed Feb. 27, 2013; which is aNational Stage of International Application No. PCT/US2013/024826 filedFeb. 6, 2013; the contents of all of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The disclosure describes cell compositions and methods for the treatmentof neurodegeneration.

BACKGROUND

Neurodegeneration is a pathological state that results in neural celldeath. Although the causes of neurodegeneration may be diverse and notalways ascertainable, a large number of neurological disorders shareneurodegeneration as a common pathological state. For example,Alzheimer's disease, Parkinson's disease, Huntington's disease, andamyotrophic lateral sclerosis (ALS) all cause chronic neurodegeneration,which is characterized by a slow, progressive neural cell death over aperiod of several years, whereas acute neurodegeneration ischaracterized by a sudden onset of neural cell death as a result ofischemia, such as stroke, or trauma, such as traumatic brain injury, oras a result of axonal transection by demyelination or trauma caused, forexample, by spinal cord injury or multiple sclerosis.

Neurodegeneration can also be triggered by a wide variety of neural cellinsults resulting from, for example, alcohol abuse, drug addiction,exposure to neurotoxins and radiation. Evidence for neurodegenerationcan even be found in dementia, epilepsy, various psychiatric disordersand as part of the normal aging process.

Regardless of the underlying cause, a growing body of evidence indicatesthat, once neurodegeneration is triggered, the outcome for all thesedisorders is invariably the same—the ultimate death of neural cells.

Stroke involves acute neurodegeneration (the rapid loss of centralnervous system neural cells with attending loss of function) due toocclusion (ischemic stroke) or rupture (hemorrhage) of a blood vesselleading to or within the brain. It usually constitutes a medicalemergency, since it can cause permanent neurological damage, systemiccomplications, and even death. Stroke is the leading cause of adultdisability in the United States and Europe and it is the number twocause of death worldwide. Stroke accounts for more than one in everyfifteen deaths in the U.S. and ranks third amongst all causes of death,behind heart disease and cancer (American Heart Association. HeartDisease and Stroke Statistics—2009 Update. Dallas, Tex.: American HeartAssociation; 2009; Rosamond et al. Heart disease and strokestatistics—2007 update: a report from the American Heart AssociationStatistics Committee and Stroke Statistics Subcommittee. Circulation.2007; 115: e69-e171). Every third stroke has a fatal outcome (Haheim etal. Risk factors of stroke incidence and mortality. A 12-year follow-upof the Oslo Study. Stroke. 1993; 24(10):1484-9). About 6% of all deathsbefore age 65 and 10% of all deaths thereafter, are due to stroke(Donnan et al. Stroke. Lancet. 2008; 371(9624):1612-23). The statisticaldata therefore demonstrate that severe disability is an unfortunate, butall too frequent, outcome for many stroke victims. Indeed, stroke is thenumber one cause of inpatient Medicare reimbursement for long-term adultcare. Total costs associated with the treatment and rehabilitation fromstroke now exceed $45 billion per year and will undoubtedly continue tocontribute to the overall increase in the cost of healthcare in the USas well as the other major industrialized nations.

Ischemic strokes are the most common form of stroke, accounting for over80 percent of all strokes. They result from arterial blockage, usuallydue to thrombosis or less commonly due to embolism. Onset is generallyabrupt and focal neurologic deficits typically ensue. About 20% ofpatients die within a few days, especially if the infarct is large.Another 10% of patients die within weeks of the initial stroke.Unfortunately, those who survive are usually severely disabled.Symptoms, depending on the area of the brain affected, includeunilateral facial or limb weakness and sensory disturbances as well ascognitive and speech impairment. The larger the area of brain affected,the more functions are likely to be impaired. Some functionalimprovement may begin to occur within days and further recovery overseveral months is common. Nevertheless, the extent of recovery isunpredictable and generally incomplete. According to the American StrokeAssociation, of those who survive a stroke, 15 to 30% are permanentlydisabled, and 20% require institutional care three months after onset(Harmsen et al. Long-term risk factors for stroke: twenty-eight years offollow-up of 7457 middle-aged men in Göteborg, Sweden. Stroke. 2006;37(7):1663-7).

Ischemic stroke leads to a core lesion, in which nerve cells die withinminutes of oxygen deprivation, and a surrounding penumbra, a region thatreceives some blood-flow and therefore some oxygen, but less thannormal. Cell death proceeds more slowly in the ischemic penumbra,typically over several hours, and is caused by variable anoxia and bytoxic substances generated by the ischemic cascade and the release ofglutamate in the core lesion. Current therapeutic interventions thusmainly target the alleviation of the injurious conditions in the strokepenumbra.

Treatments for acute ischemic stroke remain however limited.

Since brain damage occurs as a result of a reduction in blood flow tothe brain, current therapies aim to remove the arterial blockage byeither dissolving the clot (thrombolysis) or by removing the clotmechanically (thrombectomy). The faster blood flow is restored, thefewer brain cells die and the greater the chance that permanent sequelaecan be averted.

At present, only two treatments are FDA approved for stroke in theUnited States:

-   -   Recombinant tissue plasminogen activator (rt-PA; Genentech), a        drug that dissolves the arterial clot; and    -   The Merci Retrieval System (Concentric Medical Inc.) and        Penumbra System (Penumbra Inc.), a device that mechanically        removes blood clots.

All the above therapeutic approaches have major limitations.

To be effective, therapy with thrombolytic agents must be performedwithin 3 to 4.5 hours of symptom onset which means only about 3% ofpatients with acute ischemic stroke receive effective rt-PA therapy. Inaddition, thrombolytic therapies carry a substantially increased risk ofcerebral hemorrhage, which further limits their use in some individuals.

For the foregoing reasons, there is an unmet, urgent need in the art forsafe and effective therapies that mitigate and/or preventneurodegeneration, and especially neurodegeneration caused by ischemia.

SUMMARY OF THE INVENTION

Accordingly, in one embodiment, the present invention provides aheterogeneous subpopulation of bone marrow (BM) cells prepared by aprocess comprising:

-   -   a) obtaining a population of bone marrow cells from unprocessed        bone marrow;    -   b) seeding the population of bone marrow cells at a low density        on a plastic surface;    -   c) washing the seeded cell population to remove non-adherent        cells;    -   d) culturing the adherent cell population to near confluence in        serum-containing media;    -   e) serially passaging the population of cultured cells for no        more than about 7 serial passages, wherein, at each passage, the        cultured cells are seeded at a low density;    -   f) obtaining the heterogeneous subpopulation of bone marrow        cells, wherein an effective amount of the heterogeneous        subpopulation of bone marrow cells is effective at treating        neurodegeneration.

In one aspect, an effective amount of the heterogeneous subpopulation ofbone marrow cells is effective at treating neurodegeneration caused byischemia.

In one aspect, the heterogeneous subpopulation of bone marrow cells iscultured in serum containing media.

In one aspect, the population of bone marrow cells are seeded at adensity of about 10²-10⁶ cells/cm².

In one aspect, the cultured cells are seeded at a density of about 750cells/cm² or less in passaging.

The unprocessed bone marrow can be obtained from a subject who is notpre-treated with any agent that modulates cell division, for example,anti-neoplastic drugs used in chemotherapy including anti-metabolites,such as 5-fluorouracil.

In one aspect, the heterogeneous subpopulation of bone marrow cellscannot be isolated by density fractionation, e.g. a Ficoll™ or Percoll™gradient, or by ACK (Ammonium-Chloride-Potassium) lysis.

In a further aspect of the invention, the heterogeneous subpopulation ofbone marrow cells is further tested in an experimental model ofneurodegeneration caused by ischemia, wherein only those cellpopulations that demonstrate the ability to treat neurodegenerationcaused by ischemia are selected. The experimental model ofneurodegeneration caused by ischemia can be an oxygen/glucosedeprivation (OGD) cell culture model or a stroke animal model. In oneaspect, the experimental model can be an oxygen/glucose deprivation(OGD) cell culture model followed by a stroke animal model.

In another aspect, after injection into the blood stream, cells withinthe heterogeneous subpopulation migrate to a site of neurodegeneration.

In one aspect, the neurodegeneration caused by ischemia is ischemicstroke.

In another embodiment, the invention also provides a method of treatingneurodegeneration caused by ischemia, comprising injecting theheterogeneous subpopulation of bone marrow cells as described above intothe blood stream of a mammal with neurodegeneration, wherein theinjection of the heterogeneous subpopulation of bone marrow cellsreduces a neurological deficit caused by the neurodegeneration.

When the neurodegeneration is caused by ischemic stroke, the method canfurther comprise an additional treatment to increase blood flow throughan occluded blood vessel, wherein the injection of the heterogeneoussubpopulation of bone marrow cells in combination with the additionaltreatment results in a greater reduction of the neurological deficitthan after either additional treatment alone or injection of theheterogeneous subpopulation of bone marrow cells without the additionaltreatment.

In one aspect, the additional treatment to increase blood flow throughan occluded blood vessel occurs at the same time as the injection of theheterogeneous subpopulation of bone marrow cells.

In another aspect, the additional treatment to increase blood flowthrough an occluded blood vessel occurs before the injection of theheterogeneous subpopulation of bone marrow cells.

When the neurodegeneration is caused by ischemic stroke, the bloodvessel can be occluded by a blood clot. The blood clot can result fromthe rupture of an atherosclerotic plaque.

When the neurodegeneration is caused by ischemic stroke, the additionaltreatment to increase blood flow through an occluded blood vessel caninclude administering a thrombolytic agent. The thrombolytic agent canbe at least one of streptokinase, urokinase and a recombinant tissueplasminogen activator. Further, the recombinant tissue plasminogenactivator can be alteplase, reteplase, desmoteplase or tenecteplase.

In another aspect, when the neurodegeneration is caused by ischemicstroke, the additional treatment to increase blood flow through anoccluded blood vessel may include mechanical removal of a blood clotfrom the occluded blood vessel.

In a further aspect, the blood clot can be captured with a filter.

In another aspect, when the neurodegeneration is caused by ischemicstroke, the additional treatment to increase blood flow through anoccluded blood vessel may include performing an angioplasty and/orvascular stenting.

The heterogeneous subpopulation of bone marrow cells can be injectedintravenously, intra-arterially, e.g. into the carotid artery, orintra-cerebrally.

In another embodiment, the invention also provides a method for treatingneurodegeneration caused by ischemia, comprising injecting theheterogeneous subpopulation of bone marrow cells into the blood streamof a mammal with neurodegeneration caused by ischemia, wherein theinjection of the heterogeneous subpopulation of bone marrow cellsreduces the volume of an infarction caused by ischemia.

When the neurodegeneration is caused by ischemic stroke, the method canfurther comprise an additional treatment to increase blood flow throughan occluded blood vessel, wherein the injection of the heterogeneoussubpopulation of bone marrow cells in combination with the additionaltreatment results in a greater reduction of the volume of infarctionthan after either the additional treatment alone or injection of theheterogeneous subpopulation of bone marrow cells without the additionaltreatment.

In one aspect, when the neurodegeneration is caused by ischemic stroke,the additional treatment to increase blood flow through an occludedblood vessel comprises administering a thrombolytic agent. Thethrombolytic agent can be at least one of streptokinase, urokinase and arecombinant tissue plasminogen activator. Further, the recombinanttissue plasminogen activator can be alteplase, reteplase, desmoteplaseor tenecteplase.

In another aspect, when the neurodegeneration is caused by ischemicstroke, the additional treatment to increase blood flow through anoccluded blood vessel can include performing an angioplasty and/orstenting.

In another aspect, when the neurodegeneration is caused by ischemicstroke, the additional treatment to increase blood flow through anoccluded blood vessel can include mechanical removal of a blood clotnear the site of the infarction.

According to a second method, the heterogeneous subpopulation of bonemarrow cells can be injected intravenously, intra-arterially, e.g. intoa carotid artery, or intra-cerebrally.

When the neurodegeneration is caused by ischemic stroke, the inventionmay also provide a kit comprising a thrombolytic agent and theheterogeneous subpopulation of bone marrow cells as described above. Thethrombolytic agent can be at least one of streptokinase, urokinase and arecombinant tissue plasminogen activator. Further, the recombinanttissue plasminogen activator can be alteplase, reteplase ortenecteplase.

When the neurodegeneration is caused by ischemic stroke, the inventioncan further provide a kit comprising the heterogeneous subpopulation ofbone marrow cells described above and a means for mechanical removal ofa blood clot.

Additionally, when the neurodegeneration is caused by ischemic stroke,the invention can provide a composition for the treatment ofneurodegeneration caused by ischemia comprising a thrombolytic agent,the heterogeneous subpopulation of bone marrow cells described above,and a carrier for injection of the composition. The thrombolytic agentcan be at least one of streptokinase, urokinase and a recombinant tissueplasminogen activator. Further, the recombinant tissue plasminogenactivator can be alteplase, reteplase or tenecteplase.

The invention can also provide a method of producing a heterogeneoussubpopulation of bone marrow cells for the treatment ofneurodegeneration, comprising:

-   -   a) obtaining a heterogeneous population of bone marrow cells        from unprocessed bone marrow;    -   b) seeding the heterogeneous population of bone marrow cells at        a low density onto a plastic surface,    -   c) washing the seeded cell population to remove non-adherent        cells;    -   d) culturing the adherent cells from the washed population of        cells to near confluence in serum containing media;    -   e) serially passaging each of the population of cultured cells        for no more than about seven serial passages,    -   wherein, at each passage, the cultured cells are seeded at low        density, thereby obtaining the heterogeneous subpopulation of        bone marrow cells.

In one aspect, the unprocessed bone marrow described above can beobtained from a subject who is not pre-treated with any agent thatmodulates cell division, for example, anti-neoplastic drugs used inchemotherapy including anti-metabolites, such as 5-fluorouracil.

In one aspect, the heterogeneous subpopulation of bone marrow cellscannot be isolated by density fractionation, e.g. a Ficoll™ or Percoll™gradient, or by ACK (Ammonium-Chloride-Potassium) lysis.

In a further aspect of the invention, the heterogeneous subpopulation ofbone marrow cells can be further tested in an experimental model ofneurodegeneration caused by ischemia, wherein only those cellpopulations that demonstrate the ability to treat neurodegenerationcaused by ischemia are selected. Experimental models ofneurodegeneration caused by ischemia can be an oxygen/glucosedeprivation (OGD) cell culture model and a stroke animal model. In oneaspect, the experimental model can be an oxygen/glucose deprivation(OGD) cell culture model followed by a stroke animal model. In oneaspect, both models can be used to test the heterogeneous subpopulationof bone marrow cells.

In another aspect, the present invention can also provide a method ofoptimizing an experimental protocol for the isolation of a heterogeneouscell population that treats neurodegeneration caused by ischemia,comprising the steps of:

isolating a cell population according to an experimental protocol,

wherein the cell population treats neurodegeneration caused by ischemia,and

wherein the optimal parameters of each step of the protocol aredetermined by testing the effect each parameter has on the efficacy ofthe isolated cell population to treat neurodegeneration caused byischemia in an experimental model of ischemia.

In one aspect, the parameters can include cell density at seeding, cellpassage number, culture media composition or cell fractionation.

In another aspect, the experimental model of ischemia can be anoxygen/glucose deprivation (OGD) cell culture model or a stroke animalmodel, such as a middle cerebral artery occlusion (MCAO) animal model.Also both models can be used. In one aspect, the experimental model canbe an oxygen/glucose deprivation (OGD) cell culture model followed by astroke animal model.

The above-described aspects have many advantages, including the abilityof the heterogeneous subpopulation of bone marrow cells to treatneurodegeneration caused by ischemic stroke. The cell population greatlydiminishes the region of infarction and improves neurological function.

In combination with thrombolytic agents and mechanical methods of clotremoval, the cell therapy promises to make a significant clinical impacton patient survival, functioning, and quality of life after stroke.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The figures are not intended tolimit the scope of the teachings in any way.

FIGS. 1A-1K depicts the results from the screening of candidate bonemarrow-derived cell sub-populations using the in vitro OGD model (FIGS.1A, 1B, 1C, 1G, 1H, and 1I) and the in vivo MCAO rat model (FIGS. 1D,1E, 1F, 1J, and 1K).

FIGS. 1A, 1B, and 1C depict host cell survival and cytokine release(bFGF and IL-6) respectively in the in vitro OGD model in response tothe presence or absence of 7 different candidate bone marrow-derivedcell sub-populations.

FIGS. 1D, 1E, and 1F compares host cell survival, infarction volume andneurological function in the in vivo MCAO rat model in response to theNCS-01 bone marrow cell sub-population or saline injected eitherintravenously (IV) or intra-arterially (ICA).

FIGS. 1G, 1H, and 1I depicts host cell survival and cytokine release(bFGF and IL-6) in the in vitro OGD model in response to the presence orabsence of the NCS-01 bone marrow cell sub-population.

FIGS. 1J and 1K shows changes in infarction volume and neurologicalfunction in the in vivo MCAO rat model in response to the injection of3×10⁵, 10⁶ and 10⁷ NCS-01 bone marrow cells.

FIGS. 2A and 2B show changes in infarction volume (FIG. 2A) andneurological function (Panel B; as measured by the Modified BedersonScale (0=normal to 3=most severe)) in a transient (60 minutes) orpermanent MCAO rat model in response to the ICA injection of 1 ml of7.5×10⁶ NCS-01 cells or saline solution.

FIG. 3 depicts a schematic diagram that describes the protocol used inthe in vitro Oxygen Glucose Deprivation (OGD) assay.

FIGS. 4A and 4B shows the relative levels of secreted bFGF and IL-6cytokines in the cell culture media of an OGD assay after addition ofsaline, Li cells or NCS-01 cells.

FIGS. 5A, 5B, and 5C depicts host cell survival, infarction volume andneurological function in the in vivo MCAO rat model in response to aninjection of saline, Li cells and NCS-01 cells.

DETAILED DESCRIPTION

The practice of the invention employs, unless otherwise indicated,conventional molecular biological techniques within the skill of theart. Such techniques are well known to the skilled worker, and areexplained fully in the literature. See, e.g., Ausubel, et al., ed.,Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY,N.Y. (1987-2008), including all supplements; Sambrook, et al., MolecularCloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y.(1989).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart. The specification also provides definitions of terms to helpinterpret the disclosure and claims of this application. In the event adefinition is not consistent with definitions elsewhere, the definitionset forth in this application will control.

Bone marrow (BM) derived cells include a heterogeneous mixture of manydifferent types of cells. Bone marrow is composed of two main cellsystems that belong to two distinct lineages—the hematopoietic tissuesand the associated supporting stroma. Thus, at least two distinct stemcells, namely hematopoietic stem cells (HSCs) and mesenchymal stem cells(MSCs), are known to co-exist in the bone marrow (Bianco, M. Riminucci,S. Gronthos, and P. G. Robey, “Bone marrow stromal stem cells: nature,biology, and potential applications,” Stem Cells, vol. 19, no. 3, pp.180-192, 2001).

MSCs can be broadly defined both by cell-surface markers and by theirability to adhere to tissue/cell culture plastic. Thus, MSC populationsare necessarily heterogeneous, i.e. not a clonal cell population. Evenif MSC sub-populations share one or more common cell surface markers,they can nevertheless differ significantly in their biological activitydepending on the manufacturing method used to isolate them. Thisdisclosure describes a two-step screening protocol for theidentification of a heterogeneous bone marrow cell population that iseffective at treating neurodegeneration including acute onsetneurodegeneration caused by ischemia.

In a first step, bone marrow subpopulations are screened for theirability to attenuate the effect of oxygen glucose deprivation (OGD) inco-cultures comprising candidate bone marrow subpopulations and neuralcells. The activity of candidate bone marrow subpopulations to attenuateneurodegeneration is assessed by measuring the secretion of trophicfactors (bFGF and IL6) in the culture media as well as determining hostcell survival in the OGD assay.

In a second step, bone marrow derived cell populations having thestrongest activity in the OGD assay are then further screened by testingin an in vivo MCAO rat model of neurodegeneration caused by ischemia.This screening procedure facilitates the identification of aheterogeneous bone marrow subpopulation, called NCS-01, that greatlyattenuates neurodegeneration caused by ischemia, as defined herein.

Unprocessed bone marrow cells are readily available to one of ordinaryskill in the art.

As used herein, the term “unprocessed bone marrow” refers to a humanbone marrow aspirate without any additional processing, such as densityfractionation or cell sorting.

In other embodiments, “unprocessed bone marrow” is obtained fromsubjects that are not pre-treated with any agent that interferes withnormal cell growth and division, including, for example, chemotherapyagents such as anti-mitotic or anti-metabolite agents.

Examples of such agents can be found in Cancer Principles and Practiceof Oncology by V. T. Devita and S. Hellman (editors), 6th edition (Feb.15, 2001), Lippincott Williams & Wilkins Publishers.

An “anti-metabolite” agent, as used herein, relates to a compound whichinhibits or disrupts the synthesis of DNA resulting in cell death.Examples of an anti-metabolite include, but are not limited to,6-mercaptopurine; cytarabine; fludarabine; flexuridine; 5-fluorouracil;capecitabine; raltitrexed; methotrexate; cladribine; gemcitabine;gemcitabine hydrochloride; thioguanine; hydroxyurea; DNA de-methylatingagents, such as 5-azacytidine and decitabine; edatrexate; and folic acidantagonists such as, but not limited to, pemetrexed.

As used herein, the term “density fractionation” refers to well-knownlaboratory procedures for the fractionation of bone marrow cells basedon cell density using Ficoll-Paque™ or Percoll™ gradients. For example,Ficoll-Paque™ is placed at the bottom of a conical tube, and unprocessedbone marrow is then slowly layered on top of the Ficoll-Paque™. Aftercentrifugation of the cells through the Ficoll gradient, the cellsseparate into layers according to density, from top to bottom: plasmaand other constituents, a layer of mono-nuclear cells, called the buffycoat, comprising peripheral blood mononuclear cells (PBMCs) andmononuclear cells (MNCs) and erythrocytes and granulocytes in thepellet. This fractionation procedure separates erythrocytes from PBMCs.Ethylene diamine tetra-acetate (EDTA) and heparin are commonly used inconjunction with Ficoll-Paque™ to prevent clotting.

As used herein, the term “ACK lysis” refers to ammonium chloridepotassium lysis buffer (ACK lysis buffer) for lysing of red blood cellsin EDTA-anti-coagulated whole blood.

As used herein, the term “seeding the population of bone marrow cells ata low density” refers to the concentration of bone marrow cells added atthe start of cell culture. In one aspect, the bone marrow cells areseeded at a density of about 10² or about 10³ or about 10⁴ or about 10⁵or about 10⁶ cells/cm². In a preferred aspect, the bone marrow cells areseeded at a density of about 10⁵ to about 10⁶ cells/cm².

As used herein, “neurodegeneration” refers to any pathological statethat results in the progressive loss of structure or function of neuralcells, including neural cell death. Thus, neurodegeneration is apathological state caused by neurological disorders.

In one embodiment, the phrase “neural cell” includes both nerve cells(i.e., neurons, e.g., uni-, bi-, or multipolar neurons) and theirprecursors and glial cells (e.g., macroglia such as oligodendrocytes,Schwann cells, and astrocytes, or microglia) and their precursors.

In one embodiment, an “effective amount” refers to the optimal number ofcells needed to elicit a clinically significant improvement in thesymptoms and/or pathological state associated with neurodegenerationincluding slowing, stopping or reversing neurodegeneration, reducing aneurological deficit or improving a neurological response. In someembodiments, an effective amount of the NCS-01 cell population refers tothe optimal number of cells needed to reduce the volume of infarctioncaused by the sudden onset of acute neurodegeneration after a stroke. Anappropriate effective amount of the NCS-01 cell population for aparticular organism may be determined by one of ordinary skill in theart using routine experimentation.

As used herein, “treating neurodegeneration” refers to the treatment ofneurodegeneration by a NCS-01 cell population that results in aclinically significant improvement in the symptoms and/or pathologicalstate associated with neurodegeneration including slowing, stopping orreversing neurodegeneration, reducing a neurological deficit orimproving a neurological response.

As used herein, “thrombolytic agents” refers to drugs that are used inmedicine to dissolve blood clots in a procedure termed thrombolysis.Non-limiting examples of thrombolytic drugs include tissue plasminogentissue activator tPA, alteplase (Activase), reteplase (Retavase),tenecteplase (TNKase), anistreplase (Eminase), streptokinase(Kabikinase, Streptase) and urokinase (Abbokinase).

The following describes the procedures for isolating and characterizinga heterogeneous bone marrow cell sub population from whole, unprocessedbone marrow that is optimal for the treatment of neurodegenerationincluding the treatment of neurodegeneration caused by ischemia.

Isolation of Candidate Bone Marrow Cell Populations Effective forTreating Neurodegeneration Caused by Ischemia.

Whole, unprocessed bone marrow is harvested from a mammal that is notpre-treated with an anti-mitotic or anti-metabolite, such as5-fluorouracil (5-FU).

The unprocessed bone marrow is then plated directly on to tissue/cellculture plastic and expanded by serial passaging in a serum containingmedia. At each passage, cells are seeded at very low cell density, i.e.approximately 750 cells/cm² or less, and cultured to near confluencebefore additional passaging. Non-adherent cells are removed by washing.As the bone marrow is not processed by density fractionation, thestarting whole bone marrow cell population includes hematopoietic andnon-hematopoietic cells as well as both nucleated and non-nucleated bonemarrow cells.

Master (MCB) and Working Cell Banks (WCB) are then establishedpreferably at passages 3 and 5, respectively and cryopreserved.

-   When needed, WCB cells are seeded at very low density, e.g.    approximately 750 cells/cm² or less, and expanded in serum    containing media before being harvested and cryopreserved using    standard procedures.    A Two-Step Selection Protocol for Evaluating a Bone Marrow Cell    Population's Ability to Attenuate Neurodegeneration

‘Candidate’ bone marrow cell populations can then be screened using atwo-step procedure that selects the optimal bone marrow cell populationfor the treatment of neurodegeneration.

In the first step, candidate bone marrow cell populations are tested inan in vitro oxygen glucose deprivation assay where candidate bone marrowcell populations are co-cultured with neural cells under experimentalconditions that mimic neurodegeneration. Cell populations that are shownto attenuate neurodegeneration in vitro are then screened for theirability to treat neurodegeneration in vivo.

In the second step, selected candidate bone marrow cell populations areevaluated in a rat MCAO model of neurodegeneration caused by ischemia.The candidate heterogeneous bone marrow cell population demonstratingthe most activity to attenuate neurodegeneration in vivo is thenselected.

The heterogeneous bone marrow cell population selected by this two-stepscreening procedure is called the NCS-01 cell population or NCS-01.

Identification of the Optimal Cell Culture Conditions for the Isolationof a Bone Marrow Cell Population with Neurodegeneration AttenuationActivity

The two-step screening protocol is also useful in identifying theoptimal experimental conditions such as cell density at seeding, cellpassage number, culture media composition or cell fractionation for theculture of bone marrow cell populations that are able to treatneurodegeneration.

Thus, using the two screening procedure, the optimal reseeding cellconcentration is about 750 cells/cm² or less at each passage. Theoptimal culture media is a serum containing media. NCS-01 cellpopulations can be passaged for no more than about 7, or about 6 orabout 5 or about 4 or about 3 or about 2 passages from the initialplating of the whole, unprocessed bone marrow. Extended passaging i.e.beyond 7 passages from the initial plating of the whole, unprocessedbone marrow diminishes or abolishes NCS-01's ability to treatneurodegeneration in the OGD in vitro assay and in the MCAO rat model.

The OGD assay and the MCAO rat model of neurodegeneration are nowdescribed in detail below.

In Vitro Screening of Bone Marrow Cell Populations Capable of TreatingNeurodegeneration

Candidate bone marrow cell populations are first screened for theirability to prevent neurodegeneration in a neuron-astrocyte co-cultureafter culture conditions of oxygen-glucose deprivation (OGD) thatsimulate neurodegeneration caused by ischemia.

Primary mixed cultures of rat or human neurons and astrocytes are firstexposed to oxygen glucose deprivation (OGD) culture conditions (forexample, 8% oxygen, glucose free media) for about 0.5 to 3 hours toinduce neurodegeneration. Oxygen glucose deprivation of the cell cultureis then discontinued and the OGD induced neural cells are cultured underphysiological conditions for 2 hours and then co-cultured in thepresence of a candidate bone marrow cell population for an additional 3hours. Neurodegeneration is then assessed by measuring host cellviability either in the presence or absence of the candidate bone marrowcell population at 0 and from 5 hours after OGD. Host cell (neurons andastrocytes) viability can be assessed using, for example, trypan bluestaining and/or the MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.

An increase in cell viability in the presence of a candidate bone marrowcell population as compared to the cell viability of a control (withoutthe addition of the candidate bone marrow cell population) by about 5%or about 10% or about 15% or about 20% or about 25% or more indicatesthe candidate bone marrow cell population can protect and rescueneuron/astrocyte co-cultures from neurodegeneration caused by oxygen andglucose deprivation.

In addition, the culture media of neuron/astrocyte cell culturessubjected to OGD conditions in the presence or absence of candidate bonemarrow cell populations can also be assayed for the induced secretion oftrophic factors, for example, bFGF and/or IL-6 using commerciallyavailable ELISA kits.

In certain embodiments, candidate bone marrow cell populations areselected according to their ability to induce an increase in the amountof secreted bFGF and/or IL-6 in the media of neuron-astrocyteco-cultures in response to oxygen glucose deprivation, but not in theabsence of oxygen glucose deprivation.

In other embodiments candidate bone marrow cell populations are selectedaccording to their ability to induce an at least two-fold or greaterincrease in the amount of secreted bFGF and/or IL-6 in the media ofneuron-astrocyte co-cultures in response to oxygen glucose deprivation,but not in the absence of oxygen glucose deprivation.

Candidate bone marrow cell populations that decrease the incidence ofOGD-induced cell death and induce an increase in the amount of secretedtrophic factors (such as bFGF and/or IL-6) are then selected forscreening in the in vivo the MCAO rat model (see below).

For example, candidate bone marrow cell populations can be selected forfurther screening if they decrease the incidence of OGD-induced celldeath by more than about 25% and increase the amount of secreted trophicfactors by at least 10% or more.

In Vivo Screening of Candidate Bone Marrow Cell Populations Capable ofTreating Neurodegeneration

Candidate bone marrow cell populations, selected in the OGD assayscreening step described above, are tested for their ability to treatneurodegeneration in vivo. For example, the candidate bone marrow cellpopulations can be tested for their ability to treat neurodegenerationin an experimental animal model of neurodegeneration includingtransgenic models of neurodegenerative diseases (see, for example,Harvey et al. Transgenic animal models of neurodegeneration based onhuman genetic studies J Neural Transm. (2011) 118(1): 27-45; Trancikovaet al. Genetic mouse models of neurodegenerative diseases. Prog Mol BiolTransl Sci. (2011); 100:419-82; Chan et al. Generation of transgenicmonkeys with human inherited genetic disease Methods (2009) 49(1):78-84;Rockenstein et al. Transgenic animal models of neurodegenerativediseases and their application to treatment development Adv Drug DelivRev. (2007) 59(11):1093-102).

In one embodiment, the experimental animal model of neurodegenerationcan be an animal model of stroke/cerebral ischemia (review by Graham etal. Comp Med. 2004 54(5):486-96), such as the MCAO rat model, whereconstriction of a surgically implanted ligature around a cerebral arterymimics the effect of ischemic stroke by limiting the blood flow to thebrain and causes ischemia and subsequent neurodegeneration.

In the transient MCAO model, a candidate bone marrow cell population,selected in the OGD assay, is administered by constant rate infusioninto the blood stream of a transient MCAO rat. One or ordinary skill inthe art can determine suitable dosages that can range, for example, from7.5×10⁴ to 3.75×10⁷ cells. The cells can be injected into, for example,either the jugular vein (IV) or the carotid artery (ICA). Controlsconsist of administration of an equivalent volume of cryopreservationmedia or saline solution. Cells within the candidate bone marrowpopulation then migrate to the site of the infarct caused by thetransient MCAO.

Neurologic function in the presence or absence of the OGD selected bonemarrow cell populations is then evaluated using a modified BedersonNeurologic Test at various times post-infarction. The rats are thensacrificed and the volume of infarction and host cell survival aremeasured by hematoxylin and eosin (H&E) or Nissl staining of braintissue sections from treated and untreated MCAO rats. Bone marrow cellpopulations are then selected according to their ability to improveneurologic function, increase host cell survival and decrease the volumeof infarction as compared to control animals up to 28 days post-MCAO.

Combination Therapies for the Treatment of Neurodegeneration Caused byIschemic Stroke

Stopping blood flow through the middle cerebral artery for an extendedperiod of time simulates permanent arterial occlusion with a blood clot.Transient occlusion, where blood flow through the cerebral artery isstopped for a limited period of time before being restored to allowreperfusion is intended to mimic therapies such as thrombolysis ormechanical clot removal that restore blood flow to the stroke penumbraimmediately after arterial blockage caused by ischemic stroke.

In many respects, the administration of the NCS-01 cell population totransient MCAO rats simulates reperfusion of occluded arteries as aresult of thrombolysis or mechanical removal of blood clots includingangioplasty or surgical implantation of stents.

When neurodegeneration is cause by ischemic stroke, NCS-01 cellpopulation can be combined with thrombolytic therapies for the treatmentof neurodegeneration caused by ischemia. A description of thrombolyticagents and their administration can be found, for example, in U.S. Pat.Nos. 5,945,432 and 6,821,985.

Thrombolytic agents injected after an ischemic event can be administeredeither before, together or after the injection of the NCS-01 cellpopulation.

Non-limiting examples of mechanical procedures to improve blood flow toa stroke penumbra that may be used in conjunction with the injection ofthe disclosed NCS-01 cell composition include angioplasty or theimplantation of stents according to procedures that are well known inthe art.

The present invention will be described in further detail with referenceto the following examples.

EXAMPLES

The examples set forth methods for isolating, selecting and usingsubpopulations of bone marrow cells for treating neurodegenerationaccording to the present invention. It is understood that the steps ofthe methods described in these examples are not intended to be limiting.Further objectives and advantages of the present invention other thanthose set forth above will become apparent from the examples which arenot intended to limit the scope of the present invention.

Example 1 Isolation of the NCS-01 Cell Population

-   1) Isolation of Candidate Bone Marrow Cell Populations

A heterogeneous bone marrow cell population was isolated by thefollowing manufacturing process:

-   -   Human unprocessed bone marrow was harvested from pre-screened        healthy donors of 50 years of age or younger by qualified        commercial vendor. The bone marrow was harvested from a donor        that was not pretreated with any anti-mitotic agent such        5-fluorouracil.    -   The bone marrow, whether processed or unprocessed, was then        seeded at low density (10²-10⁶ cells/cm²) onto a tissue/cell        culture plastic surface and cultured in the presence of        serum-containing media;    -   after a few days to allow for cell adherence to the plastic,        non-adherent cells were removed by washing; and    -   culturing the adherent cell population to near confluence in        serum-containing media; serially passaging the population of        cultured cells for no more than about 7 serial passages,        wherein, at each passage, the cultured cells are seeded at a low        density.

To select the optimal culture conditions for the isolation of a bonemarrow cell population that can treat neurodegeneration, cellpopulations were initially grown under different culture conditions suchas cell density at seeding, cell passage number, culture mediacomposition or cell fractionation (see Table I).

Bone marrow cells from unprocessed bone marrow or after densityfractionation or ACK lysis were seeded on to tissue/cell culture plasticin presence of α-MEM supplemented with 2 mM GlutaMax (Invitrogen) and10% fetal bovine serum (FBS, HyClone or GIBCO) or α-MEM (Mediatech) with2 mM GlutaMax (Invitrogen) and 10% fetal bovine serum (FBS, HyClone orGIBCO) or serum free media (StemPro). After washing to removenon-adherent cells, adherent cells were allowed to proliferate to nearconfluence. The cells were then serially passaged for a total of 3, 4, 5or 6 passages.

Candidate bone marrow cell populations were then tested for theirability to treat neurodegeneration in the in vitro OGD assay and in vivoMCAO study.

TABLE I CELLS CELL FRACTION CULTURE MEDIA PASSAGES Cell 1 UnprocessedBM* αMEM + 10% FBS 3 Cell 2 Using Ficoll αMEM + 10% FBS 3 Cell 3 UsingACK lysis αMEM + 10% FBS 3 Cell 4 Using Ficoll Serum free media(StemPro) 6 Cell 5 Using ACK lysis Serum free media (StemPro) 5 Cell 6Using Ficoll Serum free media (StemPro) 4 Call 7 Using ACK lysis Serumfree media (StemPro) 4 *Bone marrow

-   2) Primary Screening Using the In Vitro Oxygen/Glucose Deprivation    (OGD) Protocol

Different parameters in the manufacturing process outlined above, suchas bone marrow preparation in the presence or absence of densityfractionation, seeding density, number of passages, culture media and/ortheir combination were evaluated using the in vitro oxygen/glucosedeprivation (OGD) experimental protocol to determine the optimalprocedure for the isolation of a candidate bone marrow cell populationthat can treat neurodegeneration.

The in vitro OGD model was chosen as an initial screen because it mimicsneurodegeneration caused by ischemic stroke. Specifically, OGD testedwhether or not a specific candidate bone marrow cell subpopulation canprevent neural cell death in culture and induce the secretion ofneuroprotective trophic factors, such as bFGF and IL-6.

In the in vitro oxygen glucose deprivation (OGD) model, primary mixedcultures of rat neurons and astrocytes (at a 1:1 ratio) were exposed toOGD injury (8% oxygen; glucose-free Earle's balanced salt solution) for90 minutes and returned to physiological conditions for 2 hours, afterwhich, a candidate bone marrow cell populations was added to the OGDtreated neuron-astrocyte co-culture for an additional 3 more hours.Neural cell viability was evaluated immediately after OGD and at 5 hoursafter OGD using standard trypan blue staining and MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)methodologies.

Cell Culture

Primary mixed cultures of rat neurons and astrocytes were maintained inculture following the supplier's protocol (CAMBREX, MD). Immediatelyafter thawing, cells (4×10⁴ cells/well) were seeded and grown in 96-wellplates coated with poly-lysine in Neuro basal media (GIBCO, CA)containing 2 mM L-glutamine, 2% B27 (GIBCO, CA) and 50 U/ml penicillinand streptomycin for 7-10 days at 37° C. in humidified atmospherecontaining 5% CO₂. The purity of the neuronal and astrocytic cellpopulations was then evaluated using MAP2 and GFAP immunostaining,respectively, and found to be greater than 99%.

Oxygen-Glucose Deprivation (OGD) and Co-Culture with Candidate BoneMarrow Cell Populations

Cultured cells were exposed to the OGD injury model as describedpreviously (Malagelada et al., Stroke (2004) 35(10):2396-2401) with fewmodifications. Briefly, culture medium was replaced by a glucose-freeEarle's balanced salt solution (BSS) having the following composition:116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO₄, 1 mM NaH₂PO₄, 26.2 mM NaHCO₃,0.01 mM glycine, 1.8 mM CaCl₂, and the pH was adjusted to 7.4. Culturedcells were placed in a humidified chamber to equilibrate with acontinuous flow of 92% N₂ and 8% O₂ gas for 15 minutes. Afterequilibrium was achieved, the chamber was sealed and placed in anincubator at 37° C. for 90 minutes. After this time period, OGD wasended by adding glucose to the culture medium and returning the culturesto the standard 95% O₂ and 5% CO₂ incubator. A 2-hour period of‘reperfusion’ in standard medium and normoxic conditions were thenallowed, after which, a candidate bone marrow (BM) cell population wasadded to the OGD-treated mixed neuronal-glial culture for about 3 hours.The supernatant and the bone marrow cell population were then separatedfrom the mixed culture by washing. Thereafter, cell viability andimmunocytochemistry were performed on the cells and the amounts ofsecreted trophic factors were measured using commercially availableELISA assays as described below.

Cell Viability Assays

Cell viability was evaluated at two time points: immediately after OGDand 5 hours after OGD (i.e., 2 hours of reperfusion plus 3 hours oftreatment with the selected bone marrow cell population). For thepost-OGD viability assay, the supernatant containing the bone marrowderived cells was separated from the adherent mixed neural cell culture.Trypan blue staining method was conducted and mean viable cell countswere calculated in three randomly selected areas (0.2 mm²) in each well(n=5 per treatment condition) to reveal the cell viability for eachtreatment condition. In addition, Trypan blue staining was performed onsubsets of bone marrow derived cells harvested as pellets from thesupernatant.

ELISA Assays

Trophic factors such as bFGF and IL-6 as well as possible neurotrophicfactors secreted by bone marrow-derived cells presumably participate inthe treatment of neurodegeneration simulated by the OGD cultureconditions. Thus, measuring the amount of these molecules secreted intothe culture media provides criteria by which to evaluate candidate bonemarrow cell populations that can treat neurodegeneration in vivo.Supernatants from co-cultures of neural cells and candidate bone marrowcell populations under standard culture conditions or exposed to OGDwere collected and analyzed for the presence of trophic factor secretionusing commercially available ELISA kits in accordance with themanufacturer's instructions.

The results of an OGD analysis of the bone marrow derived cellpopulations processed according to the parameters depicted in Table Iabove, are shown in FIGS. 1A, 1B, and 1C.

Based on the results depicted in FIGS. 1A, 1B, and 1C αMEM+10% FBS waschosen as the optimal cell culture medium and unprocessed bone marrowwas found to be superior to bone marrow processed by densityfractionation (such as Ficoll-Paque or Percoll) or by ACK lysis. Theoptimal passage number for unprocessed bone marrow cells in αMEM+10% FBSmedium was found to be no more than 7 passages.

-   3) Secondary Screening and Selection of Candidate Bone Marrow Cell    Populations Using the In Vivo Middle Cerebral Artery Occlusion Rat    Model

Based on the results obtained with the initial screening in the in vitroOGD model, the biological activity of each candidate bone marrow cellpopulation to treat neurodegeneration was evaluated by comparing theneurological deficit and the volume of infarction of MCAO rats treatedwith the candidate bone marrow cell population with MCAO rats treatedreceiving only saline solution.

Middle Cerebral Artery Occlusion (MCAO) Surgery

Animals were anesthetized using isoflurane (1.5%-2.5% with oxygen). Thescalp skin was shaved and scrubbed with alcohol and chlorhexidinesurgical scrub. The animal was then placed in a stereotaxic apparatus.Starting slightly behind the eyes, a midline sagittal incision about 2.5cm long was made, and the skull area was exposed using the rounded endof a spatula. With the bregma as a reference point, baseline (i.e.,prior to stroke surgery) laser Doppler recording was obtained from thefollowing coordinates (AP: +2.0, ML: ±2.0). The skin on the ventral neckwas shaved from the jaw to the manubrium and scrubbed with alcohol andchlorhexidine surgical scrub. The animal was then moved under thesurgical microscope. A skin incision was made over the right carotidartery. The external carotid was isolated and ligated as far distally aspossible. The occipital artery was cauterized. Occasionally there wasanother branch or two extending from the external carotid that may alsoneed to be cauterized. A second ligature was placed proximally on theexternal carotid artery, which was then cut between the ligatures. Thepterygopalatine artery was ligated. Following this, a temporary suturewas placed around the common carotid to provide tension and restrictblood flow. The proximal stump of the external carotid was pulled backusing the ligature, effectively straightening the carotid bifurcation.An incision using a pair of micro-scissors was made in the stump of theexternal carotid and a 4-0 nylon filament with a pre-fabricated end wasinserted and passed up into the internal carotid until resistance wasfelt (approximately 15-17 mm). This effectively blocks the middlecerebral artery (MCA). The filament was secured in place with a ligaturearound the proximal stump of the external carotid. The contralateralcommon carotid was isolated and secured with a temporary ligature. Theskin incision was closed with staples. The animal was then fixed to thestereotaxic apparatus for laser Doppler recording to reveal successfulMCA occlusion. After five minutes, the ligature to the contralateralcommon carotid artery was removed. The isoflurane was discontinued andthe animal was placed in a recovery cage over a warming blanket. After60 minutes, the animal was anesthetized again with isoflurane and theincision was opened for testing in the transient model. The filamentcausing the occlusion was removed and the stump of the external carotidligated close to the carotid bifurcation. The skin incision was closedwith staples. The animal was again fixed to the stereotaxic apparatusfor laser Doppler recording to reveal successful reperfusion. Finally,the animal was placed in a recovery cage over a warming blanket.

Neurological Function Tests

The well-recognized modified Bederson Neurologic Test was performed foreach rat and involves obtaining a score from each of the following:

-   -   contralateral hind limb retraction, which measures the ability        of the animal to replace the hind limb after it was displaced        laterally by 2-3 cm, graded from 0 (immediate replacement) to 3        (no replacement),    -   beam walking ability, graded 0 for a rat that readily traverses        a 2.4-cm-wide, 80-cm-long beam to 3 for a rat unable to stay on        the beam for 10 seconds, and    -   bilateral forepaw grasp, which measures the ability to hold onto        a 2-mm-diameter steel rod, graded 0 for a rat with normal        forepaw grasping behavior to 3 for a rat unable to grasp with        the forepaws.

The scores from all 3 tests were assessed over a period of about 15minutes on each assessment day. An average score was calculated from the3 tests to provide a composite neurologic deficit score that ranges from0 (normal neurological function) to a maximum of 3 (severe neurologicaldeficit). Thus, the higher the score, the greater the neurologicaldeficit.

Based on pilot studies, a score above about 2.5 indicates an animal hasneurological deficits characteristic of stroke.

Histology

Brain Section Preparation

Brain section preparation is designed to identify regions of cerebralinjury. At 7 days or 28 days after MCA occlusion, rats were euthanized,perfused by trans-cardiac perfusion with saline, followed by 4%paraformaldehyde. The brains were then fixed in 4% paraformaldehyde, andsubsequently immersed in 25% sucrose. Each forebrain was cut into 30 μmthick coronal tissue sections with anterior-posterior coordinatescorresponding from bregma 5.2 mm to bregma −8.8 mm per animal.

Measurements of Infarction Volume

At least 4 coronal tissue sections per brain were processed forhematoxylin and eosin (H&E) or Nissl staining. The indirect lesion area,in which the intact area of the ipsilateral hemisphere was subtractedfrom the area of the contralateral hemisphere was used to revealcerebral infarction.

The lesion volume was presented as a volume percentage of the lesioncompared to the contralateral hemisphere. Histological determination ofthe lesion volume was performed using hematoxylin and eosin (H&E) orNissl staining, with representative images captured digitally andprocessed via NIH Image J software, and quantitative image analysis. Thelesion volume was determined according to the following formula:Thickness of the section×sum of the infarction area in all brainsections.

To minimize artifacts produced by post-ischemic edema in the infarctedarea, the infarction area in the ipsilateral hemisphere was indirectlymeasured by subtracting the non-infarction area in the ipsilateralhemisphere from the total intact area of the contralateral hemisphere.

Measurements of Cell Survival in Ischemic Peri-Infarct Area

A randomly selected high power field corresponding to the corticalperi-infarct area was used to count cells surviving in this ischemicregion (Yasuhara et al., Stem Cells and Dev, 2009). For an estimation ofhost neuronal cell viability within the ischemic cortical region, Nisslstaining was performed using Crystal violet solution (Sigma, St. Louis,Mo.), and randomly selected visual fields of the cortical region andcorresponding contralateral intact cortex in 3 sections were capturedphotographically (Carl Zeiss, Axiophot2), and cell numbers weredetermined by counting cells in randomly selected high power field views(28,800 μm2). The percentages of preserved neurons in damaged cortexrelative to the intact side were calculated and used for statisticalanalyses. Brain sections were blind-coded and the total number ofcounted stained cells was corrected using the Abercrombie formula.

Screening of Candidate Bone Marrow Cell Populations Using the MCAOStroke Animal Model

Groups of 3 (for IV administration) or 6 (for ICA administration) malerats/group were subjected to 1 hr. transient MCAO and then injected with1 ml of injection medium containing either saline or 7.5×10⁶ bonemarrow-derived cells (called NCS-01 cell population) isolated accordingto the protocol described above. Animals were followed for up to 7 dayspost cell administration.

FIGS. 1D, 1E and 1F shows that IV or ICA-administered NCS-01 bone marrowcell population provided substantial neurologic and pathologic benefit,when administered in rats with transient MCAO. Moreover, NCS-01prevented host cell death by treating ischemia-induced neurodegenerationwith consequent reduction in infarction volume and improvement ofneurological deficit.

The primary in vitro and secondary in vivo screening procedures wererepeated until the process reliably and reproducibly produced an optimalsubpopulation of bone marrow derived cells (called NCS-01 cellpopulation) that was able to treat neurodegeneration.

The optimized NCS-01 cell population was again tested in the in vitroOGD model to confirm anti-neurodegeneration activity in a co-culture ofhuman neurons and astrocytes (see FIGS. 1G, 1H and 1I) and in the invivo rat MCAO model (FIGS. 1J and 1K). The experiment depicted in FIGS.1J and 1K also shows the ability of NCS-01 cell population to treatneurodegeneration is dose dependent.

Example 2 Standardized Manufacturing Procedure for the Product of theNCS-01 Cell Population that is Able to Treat Neurodegeneration

Whole, unprocessed bone marrow is harvested from a mammal that is notpre-treated with any anti-mitotic or anti-metaolite agent, such as5-fluorouracil (5-FU). Because the bone marrow is not processed bydensity fractionation or ACK lysis, the starting whole bone marrow cellpopulation can include hematopoietic and non-hematopoietic cells as wellas both nucleated and non-nucleated bone marrow cells.

The above unprocessed bone marrow is diluted and seeded at low density(105-106 cells/cm2) onto a tissue/cell culture plastic surface andcultured in the presence of serum-containing media (α-MEM (phenol redfree) supplemented with 2 mM GlutaMax (Invitrogen) and 10% fetal bovineserum (FBS, HyClone or GIBCO) or α-MEM (Mediatech) with 2 mM GlutaMax(Invitrogen) and 10% fetal bovine serum (FBS, HyClone or GIBCO)). Thecell cultures are then incubated at 37° C., 5% CO₂, 80% RH (relativehumidity) for 72 hours;

The cells are rinsed with D-PBS to remove non-adherent cells and RBCsfrom the cell culture plastic followed by a complete medium exchangewith supplemented α-MEM, which is used for all subsequent feedings. Cellcultures (at passage 0 or p0) were then incubated at 37° C., 5% CO2, 80%RH.

For passage 1, the cells are rinsed with D-PBS and the plastic adherentcells are detached using a cell dissociation reagent. Dissociated cellsare harvested by centrifugation at 300 g (˜1000 rpm) for 8-10 minutes.The pelleted cells are re-suspended with supplemented α-MEM and seededat a density of approximately 750 cells/cm² onto a tissue/cell cultureplastic surface.

The cells are cultured to near confluence before additional passaging.

Cells are harvested and again seeded again at a density of approximately750 cells/cm² onto a tissue/cell culture plastic surface as describedabove for passage 2.

For passage 3, cells are harvested using a cell dissociation reagent andcentrifuged as described above. The pelleted cells are then resuspendedand pooled. Cells are resuspended in a cryopreservation media and 1 mLcell suspension aliquoted into Cryovials (Nunc). Vials are frozen usinga control-rate freezer and once the freezing program is complete,transferred on dry ice for permanent storage in a vapor-phase LiquidNitrogen freezer.

The vials containing cells at passage 3 (p3) constitute the MCB.

One vial of MCB is thawed and the recovered cells (passage 4 or P4) areseeded on to tissue/cell culture plastic at a density of approximately750 cells/cm² in α-MEM supplemented with 10% FBS and GlutaMAX™. Thecells are then incubated at 37° C., 5% CO₂, 80% RH.

For passage 4, the cells are cultured to near confluence beforeadditional passaging. Cells are harvested and seeded onto newtissue/cell culture plastic surface as described above.

For passage 5, cells are harvested and frozen, following the methodsdescribed above for the MCB. Cells that are aliquoted into vials andcryopreserved at passage 5 (P5) constitute the WCB.

When needed, one vial of WCB is thawed and the recovered cells areseeded on to tissue/cell culture plastic at a density of approximately750 cells/cm² in α-MEM supplemented with 10% FBS and GlutaMAX™. Thecells are then incubated at 37° C., 5% CO₂, 80% RH.

For passage 6, the cells are cultured to near confluence beforeadditional passaging. Cells are harvested and seeded onto newtissue/cell culture plastic surface as described above.

Culture media is changed in the middle of passage (WCB to passage 6 andpassage 6 and passage 7).

For passage 7, cells are harvested and frozen, following the methodsdescribed above for the MCB.

Example 3 Treatment of Neurodegeneration Caused by Permanent VersusTransient Middle Cerebral Artery Occlusion (MCAO) with NCS-01 Cells

The infarction volume and neurologic function of NCS-01 treated ratswith permanent MCAO was compared to that of NCS-01 treated rats withtransient (60 minutes) MCAO.

The transient MCAO simulates the treatment of arterial occlusion causedby stroke with now standard clinical procedures that restore blood flowto the stroke penumbra. These procedures include the administration ofthrombolytic drugs as well as procedures such as angioplasty and/orvascular stenting that involve the mechanical removal of blood clots.

Groups of 3 or 6 rats were subjected to either permanent or transientMCAO (with reperfusion). Either saline or 7.5×10⁶ NCS-01 cells in 1 mlwere then administered ICA 24 hours post-ischemia and rats weremonitored for up to 28 days. Neurologic function was then evaluatedusing the modified Bederson Neurologic Test. The transient occlusionmodel mimics the situation in which a stroke patient was treated withtPA, or was subjected to clot removal.

The results in FIGS. 2A and 2B show significant histological (infarctionvolume; FIG. 2A) and clinical (modified Bederson Scale; FIG. 2B) benefitwith NCS-01 cell population treatment in both MCAO paradigms. Benefitwas 2- to 3-fold greater in the transient occlusion model than in thepermanent occlusion model. The time course of the neurological response(Panel B) showed steady improvement up to 28 days post-infarction,averaging 11% over this interval for un-reperfused (permanent occlusion)and untreated controls to 67% for re-perfused (transient occlusion) andNCS-01 treated animals.

Unexpectedly, NCS-01 was more effective in treating symptoms in thetransient occlusion model, suggesting that maximum efficacy may beobtained when NCS-01 was added in conjunction with clot removal, eitherafter administration of a thrombolytic drug or removal of the clot usinga mechanical device.

Example 4 Comparison Between NCS-01 Cells and Other Bone Marrow DerivedCells

-   1) Isolation of a Bone Marrow Cell Population According to Li et al.

A bone marrow cell population was prepared exactly as described in thepublication by Li et al. Journal of Cerebral Blood Flow and Metabolism(2000) 20: 131 1-1319 (hereafter called the Li bone marrow population).

Primary cultured bone marrow cells were obtained from adult mice thathad received the anti-metabolite drug, 5-fluorouracil (5-FU, 150 mg/kg)intra-peritoneally 2 days before harvesting (Randall and Weissman,1997). Using a 21-gauge needle connected to a 1 mL syringe withphosphate-buffered saline (PBS, 0.5 mL), fresh complete bone marrow washarvested aseptically from tibias and femurs. Bone marrow wasmechanically dissociated until a milky homogenous single-cellsuspension. Red blood cells were removed from bone marrow using 0.84%NH₄Cl and the number of nucleated marrow cells was determined using acytometer. 2×10⁶ nucleated cells were seeded into a tissue culture flaskin Iscove's Modified Dulbecco's medium supplemented with fetal bovineserum (10%). After 3 days of incubation, cells tightly adhered toplastic and were resuspended in fresh Iscove's Modified Dulbecco'smedium in new flasks and were grown for another three passages.

-   2) Comparison of the Li Cell Population with the NCS-01 Cell    Population in the In Vitro OGD Assay

Two lots of NCS-01 manufactured from different WCBs of a same MCB andbone marrow were tested together with the Li cell population in the invitro OGD model as outlined in FIG. 3.

As shown in FIGS. 4A and 4B, both lots of NCS-01 cells generated thesame increase in the secretion of both IL-6 and bFGF. Hence, NCS-01 cellpopulations produced by the optimized manufacturing procedure describedabove consistently treated neurodegeneration in the in vitro OGD assay.

In contrast, the Li cell population, that was isolated exactly asdescribed in the Li (2000) publication, produced 4-5× less bFGF and IL-6than NCS-01 cell population in the OGD assay.

-   2) Comparison of the Li Cell Population with the NCS-01 Cell    Population in the In Vivo MCAO Assay

The ability of the NCS-01 and Li cell populations to treatneurodegeneration in vivo was tested in vivo using the MCAO rat model.The effect of the cells on host cell viability, the volume of infarctionand neurological deficit are shown in FIGS. 5A and 5B. Consistent withthe studies described above, the NCS-01 cell population prevented hostcell death (see FIG. 5A) by treating ischemia-induced neurodegenerationwith consequent reduction in infarction volume and improvement ofneurological deficit (see FIGS. 5B and 5C).

In contrast, the Li cell population failed to show any statisticallysignificant activity on infarction volume or neurological function.These data therefore demonstrate that the ability of the NCS-01population to treat neurodegeneration is defined by the manufactureprocess and that the NCS-01 cell population is distinct from the cellpopulation described in the Li 2000 publication.

Any patent, patent application, publication, or other disclosurematerial identified in the specification is hereby incorporated byreference herein in its entirety. Any material, or portion thereof, thatis said to be incorporated by reference herein, but which conflicts withexisting definitions, statements, or other disclosure material set forthherein is only incorporated to the extent that no conflict arisesbetween that incorporated material and the present disclosure material.

The invention claimed is:
 1. A method of producing a heterogeneoussubpopulation of bone marrow cells for the treatment ofneurodegeneration caused by ischemia, comprising: a) obtaining apopulation of bone marrow cells from human unprocessed bone marrow; b)seeding the population of bone marrow cells at a low density of 10⁵-10⁶cells/cm² onto a plastic surface, c) washing the seeded cell populationto remove non-adherent cells; d) culturing the adherent cells from thewashed population of cells to near confluence in serum containing media;e) serially passaging the cultured cells for no more than seven serialpassages in the serum-containing media, wherein, at each passage, thecultured cells are seeded at low density of about 750 cells/cm²; f)obtaining said heterogeneous subpopulation of bone marrow cells; and g)testing said heterogeneous subpopulation of bone marrow cells in anexperimental in vitro and/or in vivo model of neurodegeneration causedby ischemia, and selecting only those populations that demonstrate theability to treat neurodegeneration in said model.
 2. The methodaccording to claim 1, wherein the serum-containing media are bovineserum-containing media.
 3. The method according to claim 1 or 2, whereinsaid unprocessed bone marrow is obtained from a subject that is notpre-treated with a chemotherapy agent.
 4. The method according to claim3, wherein said chemotherapy agent is 5-fluorouracil.
 5. The methodaccording to claim 3, wherein said population of bone marrow cellscannot be isolated by density fractionation or by ACK lysis.
 6. Themethod according to claim 5, wherein said density fractionation uses adensity gradient medium comprising a co-polymer of sucrose andepichlorohydrin or polyvinylpyrrolidone-coated silica.
 7. The methodaccording to claim 1, wherein the experimental in vitro model ofneurodegeneration caused by ischemia is an oxygen/glucose deprivation(OGD) cell culture model.
 8. The method according to claim 1, whereinthe experimental in vivo model of neurodegeneration caused by ischemiais a stroke animal model.
 9. A method of optimizing an experimentalprotocol for the isolation of a cell population that treatsneurodegeneration caused by ischemia, comprising the steps of: isolatinga cell population according to the method according to claim 1, anddetermining the optimal parameters of each step of the method accordingto claim 1 by testing the effect each parameter has on the efficacy ofsaid isolated cell population to treat neurodegeneration caused byischemia in the experimental in vitro and/or in vivo model ofneurodegeneration caused by ischemia.
 10. The method according to claim9, wherein said neurodegeneration caused by ischemia is caused byischemic stroke.
 11. The method according to claim 9, wherein saidparameters comprise cell density at seeding, cell passage number,culture media composition and/or cell fractionation.
 12. The methodaccording to claim 9, wherein said experimental in vitro model ofneurodegeneration caused by ischemia is an oxygen/glucose deprivation(OGD) cell culture model.
 13. The method according to claim 9, whereinsaid experimental in vivo model of neurodegeneration caused by ischemiais a stroke animal model.
 14. The method according to claim 9, whereinsaid experimental in vivo model of neurodegeneration caused by ischemiais a middle cerebral artery occlusion (MCAO) animal model.